Adenylate Cyclase in hepatic membranes is regulated by hormones, guanine nucleotides, and divalent cations. However, when solubilized with either neutral or ionic detergents, the enzyme loses activity when assayed in the presence of magnesium ions, guanine nucleotides, and hormones. When the enzyme is pre-activated with GMP-PNP prior to solubilization, the enzyme retains divalent cation regulation and most of the activity is recovered. Detergent solubilization of the nonpreactivated enzyme appears to result in dissociation of the catalytic unit from components involved in metal ion, guanine nucleotide, and hormonal regulation. The dissociated enzyme uses MnATP rather than MgATP as substrate. The selective use of MnATP as substrate is also a characteristic of the normally soluble adenylate cyclase in rat testis.